Exfoliative Cytopathology

Ma. Minda Luz M. Manuguid, M.D.

Exfoliative Cytopathology

• def : study of cells exfoliated (naturally or artificially) or aspirated (FNAB) from body surfaces or cavities

• staining: Papanicoulaou (Pap) method• primary objective: detect neoplastic cells • other purposes:

identify infectious organisms &/or their products; detect inflammatory conditions; assess hormonal status; diagnose other disorders

Exfoliative Cytopathology

• Advantages: Can assess large areas/tissues Non-invasive/ minimally invasive Can be done repeatedly Low cost Rapid processing & evaluation

• Disadvantages: Cannot localize lesion Needs to be confirmed by histopathology Inter-observer variation greater than with histopathology

Cytopathology specimens

• Cervicovaginal smear – most common – “Pap” smear

• Buccal scrape• Pleural fluid• Pericardial fluid• Peritoneal/Ascitic fluid• GIT secretions: gastric juice, intestinal juice, • RT secretions: bronchial lavage; pharyngeal;• GUT secretions: bladder lavage; urethral

secretions;• Fine Needle Aspiration Biopsy

Papanicoulaou staining

• 70% Ethanol 2 min• Water 2 min • Harris Hematoxylin 4 min• HCl 0.05% 1min 30sec / Water 5 min• 95% Ethanol 2 min• Orange G 3 min• 95% ethanol 2 min• EA 50 4 min• 95% ethanol 2 min• Absolute Ethanol at least 2 min, 2

changes

Barr body

Cytology specimens

Urine cytology

Sputum cytology bronchial lavage

FNA, lymph node

Pleural effusion cytology

Benign conditions

Liver aspirateThyroid aspirate

fatty Liver aspiratesialadenitis

Malignancies

Papillary adenoCA, thyroid

Ascitic fluid

Mucinous CA from Ovary

Pleural fluid

Malignant mesothelioma

Mucinous adenoCA, breast

Papillary CA, thyroidD

Malignancies

Lymphoma, lymph node aspirate

Small cell Lung CA

Transitional cell carcinoma

Gastric cancer

CervicoVaginal Cytopathology

• specimen: smears from the vaginal canal (lateral vaginal wall if cytohormonal index is required), vaginal pool (posterior cul-de-sac), ectocervix, transformation zone / endocervix

• purpose: detect Cancer or the cellular changes that can lead to Cancer (“early detection”)

• recommended timing: Baseline smears at age 18 or age at first sexual intercourse Yearly during reproductive age plus when pregnant or with gynecologic

symptoms

Slide prep (Conventional)

• 1 - Smearing of the exocervical sample with a wooden spatula (Ayre's spatula). Some may have a longer bifid extremity for a better endocervical sampling.

• 2 - Smearing of the endocervical sample taken with the thinner extremity of the wooden spatula.

• 3 - Spray fixation: immediate, during a few seconds, with a spray/slide distance around 20 cm.

Cytobrushes

• Different types of brushes allowing to collect cells from the ectocervix and endocervix. These brushes can be used for conventional smears instead of the Ayre’s spatula. They are mandatory for liquid based preparation.

Liquid based (thin) prep

• Head of spatula, where cells are lodged, is broken off into small glass vial containing preservative fluid, or rinsed directly into preservative fluid

• Sample is sent to lab, then spun and treated to remove mucus, pus or other obscuring material

• Random sample of remaining cells is taken and deposited onto a slide

• Reduces number of inadequate smears and need for repeat smears

• Thin-Prep appears to be superior to convention Pap test in detecting SIL

Reporting : Bethesda 2001

• Specimen Source:

• vaginal pool / lateral vaginal wall; • ectocervical; • transformation zone / endocervical

Type: • conventional; • liquid-based (thin) prep; • other : e.g. air-dried, diff quick

Reporting: Bethesda 2001• Specimen adequacy

Satisfactory: • Cellularity: conventional = 8000-12000, liquidbased = 5000; • transformation zone component: 10 well-preserved

endocervical or metaplastic cells Unsatisfactory

• Specimen rejected/not processed: (not labeled; broken slide; unacceptable method of transport;

• Specimen processed and examined, but unsatisfactory for evaluation of epithelial abnormality: extensive (>75%) obscuring inflammation / hemorrhage; cytolysis; drying artifact;

Adequate smear

• Satisfactory Specimen: Adequate cellularity & transformation zone component: superficial and intermediate squamous cells and a cluster of columnar endocervical cells.

Lactobacilli• Lactobacilli and occasional

cytolysis. Bacteria get more dispersed in liquid preparations; the background is thus cleaner. (contrast with lower right inset from a conventional smear).

• Lactobacilli (Doderlein bacilli) are normal flora

• In determining specimen adequacy, nuclear preservation and visualization are of key importance. Changes such as cytolysis and partial obscuring of cytoplasmic detail may not necessarily interfere with specimen evaluation. Abundant cytolysis(>~50%) may be mentioned as a quality indicator, but most such specimens do not qualify as unsatisfactory unless nearly all of the nuclei are devoid of cytoplasm.

Liquid-based Conventional

Obscuring factors

inflammation / WBCs

hemorrhage / RBCs

drying artifact

“Cornflakes” artifact

• Also called “brown artifact”, cornflaking

• Distinctive appearance is due to evaporation of xylene before cover slipping, with deposition of air on superficial squamous cells

• More common on conventional than liquid based preparations

CytoHormonal Maturation Index

• specimen: middle third segment, lateral vaginal wall sample

• adequate cellularity: at least 300 cells• purpose: assess hormonal status indirectly by

determining the differential of the squamous lining cells expressed as percentages (ratio of P : I : S cells)

• P / I / S : parabasal/intermediate/superficial

P I S

Newborn (Maternal hormones present) 0 80 20

Childhood, pre-puberty 100 0 0

Pre-ovulatory (Estrogen dominant) 0 30 70

Post-ovulatory (Progesterone dominant)

0 60 40

Pregnancy (Progesterone dominant) 0 100 0

Menopause 100 0 0

Bethesda 2001: Non-neoplastic findings

• Organisms Trichomonas vaginalis (may be with Leptothrix) Fungal organisms morphologically consistent with Candida spp. Shift in flora suggestive of bacterial vaginosis (Gardnerella

vaginalis) Bacteria morphology consistent with Actinomyces spp. Cellular changes consistent with Herpes simplex virus

Trichomonas vaginalis

• pear-shaped, flagellated, w/ blue-green cytoplasm, ovoid eccentric nucleus

• Frequently associated with Leptothrix (long slender bacteria): “spaghetti & meatballs”

• Squamous cell changes in Trichomoniasis Minimal nuclear enlargement Cytoplasmic polychromasia

Trichomonas and Leptothrix• The finding of

Trichomonas and Leptothrix together has been referred to as "spaghetti and meatballs" When Leptothrix are seen, one should search for the possible presence of trichomonads. In liquid based preparations, the leptothrix organisms may tend to clump (arrow) as opposed to conventional smears

Candida albicans • Candida: yeasts & pseudohyphae formed by

elongated budding, with spearing of epithelial cells--"shish kebab" effect.

• predisposing factors: diabetes mellitus; pregnancy; change in pH of vaginal secretions / loss of normal flora (Lactobacilli)

Bacterial vaginosis

• Gardnerella vaginalis – coccobacilli – proliferate when vaginal pH becomes less acidic & normal flora is eradicated

• Clue cells – individual squamous cells covered by a layer of bacteria that obscures the cell membrane. Background is usually clear in liquid based preparations.

Clue cell

Actinomyces spp. • Gram-positive, thin

filamentous bacilli in tangled clumps, often with acute angle branching, sometimes showing irregular “wooly” appearance. Swollen filaments may be seen with clubs at periphery.

• A “cotton ball”- like acute inflammatory response is common.

• often associated with intrauterine device (IUD) usage. Organisms may alert clinician to look for evidence of pelvic infection.

Herpes • Multinucleation, Nuclei showing

"ground-glass" appearance due to intranuclear viral particles

• nuclear Molding, and • enhancement of nuclear

envelope caused by peripheral chromatin Margination.

• Cowdry type inclusions – intranuclear dense eosinophilic amorphous or droplet-like bodies surrounded by a halo

Chlamydia trachomatis• an obligate intracellular parasite

with elementary bodies (infectious but incapable of cell division) and reticulate bodies (multiply within cytoplasm, but not infectious until they transfer back into elementary bodies)

• Cytology: morphologic changes (intracytoplasmic inclusions with central small coccoid bodies) are not specific: mixed flora and many neutrophils;

• If clue cells, yeasts and trichomonads, are absent on a Gram stain and a wet mount, Chlamydia should be suspected

• Presence of infection may not be associated with symptoms

• Diagnosis is based on molecular tests (PCR or ligase chain reaction)

Non-neoplastic findings

• Atrophy• Glandular cells status post hysterectomy• Reactive cellular changes associated with

Inflammation (including typical repair) Radiation Intrauterine contraceptive device

• Others Tubal metaplasia Keratotic cellular changes Lymphocytic (follicular) cervicitis other

Atrophic vaginitis

• Parabasal cells with mostly bland nuclei (some showing air drying). Some degenerated cells with pyknosis also present. Basophilic granular background with inflammation also present.

Repair• Repair is characterized by

cohesive cell groups “monolayer sheets” with distinct cellular outlines, nuclei oriented in the same direction (streaming), increased nuclear size and prominent nucleoli. There are few or no single cells.

• Absence of single cells with nuclear changes and lack of marked anisonucleosis or irregularities in chromatin distribution or variation in size and shape of nuclei indicates this is typical repair (as opposed to "atypical repair")

Non-neoplastic findings

Glandular cells post-hysterectomy

Radiation effects IUD effects

Tubal metaplasia

parakeratosis

hyperkeratosis

Squamous metaplasia

• Normal polygonal squamous metaplastic cells with round to oval nuclei and bland chromatin pattern.

• Change from columnar to squamous lining cells – signifies chronic cervicitis

• The presence of squamous metaplastic cells indicates that the transformation zone has been sampled (a minimum of 10 well-preserved endocervical or metaplastic cells is required for this quality indicator).

Reporting: Bethesda 2001

• Endometrial cells in a woman aged 40 years or older

• Atypical Squamous cells Of undetermined significance (ASCUS) Cannot exclude HSIL (ASC-H)

• Epithelial abnormalities (squamous) Low-grade squamous intra-epithelial lesion (LSIL) encompassing

HPV, mild dysplasia, CIN 1 High-grade squamous intraepithelial lesion (HSIL) encompassing

moderate & severe dysplasia, CIN 2, CIN 3, CIS• With features suspicious for invasion

Endometrial cells

• Endometrial cells after age 40, particularly out of phase or after menopause may be associated with benign endometrium, hormonal alterations and less commonly, endometrial /uterine abnormalities (rarely, Endometrial CA)

• Because of this association, all glandular endometrial cells are reported in women 40 years and over.

Menstrual smear

Endometrial cells

ASCUS

• mild nuclear enlargement, binucleation• hyperchromasia, fine chromatin• smooth nuclear & cytoplasmic

membranes• no clear-cut evidence for diagnosis of an

intraepithelial lesion

ASC – H

• Less mature squamous cells/metaplastic cells with polygonal shape, and slightly enlarged nuclei with occasional nuclear contour irregularities.

• Atypical multinucleated cells.

LSIL - HPV

• Mature squamous cells displaying enlarged nuclei (3 to 4 times the size of normal intermediate cell nuclei) with granular chromatin

• Binucleation and koilocytosis are consistent with HPV cytopathic effect.

HSIL

• Metaplastic cells with increased N:C ratios and nuclear contour irregularities.

• Hyperchromasia, abnormally distributed chromatin.

• dense / "metaplastic" cytoplasm

HSIL r/o Invasion

• Numerous abnormal cells are present in varying sizes and shapes. Nuclei vary from round to oval to spindle shaped. This type of pleomorphism suggests invasive carcinoma. However, the absence of nucleoli and necrosis are consistent with CIS/HSIL

• Keratinized dysplastic cells with nucleoli, and angulated or carrot-shaped nuclei that may raise suspicion for invasion and qualify for an interpretation of HSIL, cannot rule out invasion. 

Reporting: Bethesda 2001

• Epithelial abnormalities (Squamous) Squamous cell carcinoma Others

• Keratinizing lesions• Squamous intraepithelial lesions

(SIL) – borderline• SIL with Glandular involvement

• Epithelial abnormalities (Glandular) Atypical, NOS / specify in

comments• Endocervical / Endometrial /

Glandular Atypical, favor Neoplastic

• Endocervical / Endometrial / Glandular

Endocervical adenoCA in situ Adenocarcinoma

• Endocervical / Endometrial / Extrauterine

• Not otherwise specified (NOS)

Invasive Squamous Cell Carcinoma

• Tumor diathesis (lysed blood and stripped nuclei); variation in cell size and shape; evidence of keratinization; scant cytoplasm; nuclei with irregularly distributed, coarsely granular chromatin and prominent nucleoli; ragged cellular borders

• Dysplastic squamous cells with anisocytosis and anisonucleosis including keratinization and tadpole cells are diagnostic of invasive squamous cell carcinoma.

• centrally located nuclei and flat arrangement of cells is consistent with squamous cell carcinoma.

Epithelial Abnormalities

HSIL & Endocervical AdenoCA Endocervical adenoCA

Endometrial adenoCarcinoma

•Irregular chromatin distribution and prominent or macronucleoli are classic findings in invasive endocervical adenocarcinoma.

Loose aggregate of small epithelial cells with slightly enlarged nuclei, small nucleoli, and vacuolated cytoplasm; "watery diathesis" and histiocytes

Extrauterine (Metastatic) Malignancies

• Colonic adenoCA: atypical glandular cells with nuclear pleomorphism, hyperchromasia, and cellular overlap; tall columnar cells and scattered goblet cells with distended mucin-filled vacuoles

• Malignant Melanoma: brown cytoplasmic pigment consistent with melanin in the malignant cells - Large single or loosely cohesive cells with round hyperchromatic nuclei, irregular nuclear membranes, coarsely clumped irregularly distributed chromatin and prominent nucleoli

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